In this study, the pesticide sensor by graphene quantum dots (GQDs) and active enzyme, GQDs/enzyme platform, has been developed for monitoring the organophosphate pesticides. In this concept, H2O2 generated from the active enzymatic reaction of acetylcholinesterase (AChE) and choline oxidase (CHOx) enables to react with GQDs resulting in a “turn-off” photoluminescence of GQDs. A “turn-on” photoluminescence of GQDs at 467 nm was recovered in the presence of organophosphate. Definitely, the photoluminescence changes of GQDs/AChE/CHOx biosensor reasonably correspond to the amount of pesticide. The detection limit of GQDs/AChE/CHOx biosensor towards dichlorvos was 0.172 ppm (0.778 μM). In this approach, this biosensor offers the promising determination of the organophosphate pesticides and a benefit for easy checking of organophosphate pesticides in food, water and environment with low cost, easy-to-prepare and less toxic to environment.